RAPD technique is a useful tool to distinguish Penicillium species.

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate genetic diversity among 13 soil Penicillium strains originating from widely dispersed areas. Twenty one of the 34 synthetic random primers were found to identify polymorphism in amplification products. The results show a high level of diversity of RAPD markers among the strains. All the strains could be identified by their characteristic amplification profile, using selected random primers. This suggests that RAPD analysis is a useful and reliable assay for characterizing the species of Penicillium genus.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

不同来源鼠李糖乳杆菌的随机扩增多态DNA分析

[目的]建立鼠李糖乳杆菌(Lactobacillus rhamnosus,Lr)菌株之间的分子鉴别方法并分析不同分离株之间的遗传多样性.[方法]从56份采集自中国新疆和田和广西巴马瑶族自治县的长寿老人粪便样本中分离得到的乳酸菌中,经生理生化分析和API 50CHL试验条鉴定,获得10株Lr.对10株Lr分离株和1株Lr标准株ATCC7469进行了随机扩增多态DNA分析,从50条随机引物中筛选到5条在菌株水平上具有鉴别力的引物P14、OPG28、OPG25、P7和P4并建立和优化了Lr菌株RAPD指纹图谱扩增方法.根据RAPD结果计算菌株间的遗传相似系数并进行聚类分析.[结果]获得了清晰稳定的DNA指纹图谱,扩增产物大小在100～2000bp之间,菌株间呈现显著的DNA多态性,不同来源的Lr分离株的遗传相似系数在0.581～0.935之间,在相似系数0.80水平上可以将11株Lr菌株分为5个类群,其中分离自新疆和田的Lr菌株归在类群B和类群C,而分离自广西巴马瑶族自治县的Lr菌株归在类群D和类群E.[结论]应用RAPD方法对Lr菌株进行分子鉴别是可行的,不同来源的Lr之间存在着较大的种内遗传多态性和不同的亲缘关系.     

有性生殖对栗疫病菌群体结构的影响

采用RAPD方法对来源于栗疫病菌8个不同子囊壳的子囊孢子后代和无性生殖的对照群体各23个菌株进行了群体结构的比较。从RAPD随机引物中筛选出扩增多态性丰富的4条引物,共扩增出条带73条,多态性检测率为100%。研究结果表明,在8个子囊壳和无性生殖群体中的基因多样性,64.27%由群体内部引起,只有35.73%的多样性由群体之间的基因差异引起。各子囊壳群体间存在的基因流动很小(Nm=0.8994)。有性群体和无性群体之间的遗传距离为0.1389,基因流动值为3.4212,说明子囊壳群体和无性生殖群体之间存在一定的系统关系。分析表明栗疫病菌子囊孢子后代在自然界的传播对自然界的病菌的多样性起重要的作用。     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Detection of genetic polymorphism by RAPD-PCR among isolates of Bacillus thuringiensis.

Sixteen isolates of Bacillus thuringiensis recovered from different Jordanian habitats were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the molecular level. Total genomic DNA from each isolate and three reference strains were amplified using 10-mer primers. Electrophoretic analysis of the amplification products revealed the incidence of polymorphism among the isolates. Pair-wise comparisons of polymorphic products were used to construct a dendrogram applying the cluster analysis. Fifteen of the isolates were all in one major cluster which was divided into six small groups. Such analysis showed some regional variation among the isolates, but did not indicate a clearly defined habitat locational pattern of the DNA polymorphism.     

Analysis of Genetic Diversity of Fusarium oxysporum f. sp. phaseoli Isolates, Pathogenic and Non-pathogenic to Common Bean (Phaseolus vulgaris L.)

Twenty isolates of Fusarium oxysporum from Brazil, pathogenic and non‐pathogenic to common bean, were analysed using random amplified polymorphic DNA (RAPDs) to study the genetic diversity. RAPD analysis using 23 oligonucleotides resulted in the amplification of 229 polymorphic and 7 monomorphic DNA fragments ranging from 234 to 2590 bp. High genetic variability was observed among the isolates, with the distances varying between 8% and 76% among pathogenic, 2% and 63% among the non‐pathogenic and 45% and 76% between pathogenic and non‐pathogenic isolates. The analysis of genetic distance data showed that the pathogenic isolates tended to group in one group and the non‐pathogenic in another. The genetic distance values of 30% among the pathogenic isolates in cluster A are compatible with the genetic distance values observed within the physiological races, but the distance values among the pathogenic isolates in clusters B and G are not compatible with the distance values observed within the race. Although our results are preliminary, it was not possible to exclude the existence of more than one race of this fungus in Brazil.     

中国棉花枯萎菌生理小种的RAPD分析*

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）分子标记方法对我国棉花枯萎菌３个生理小种（３、７、８号）进行遗传多样性分析,以筛选出的１０个随机引物对采自我国１１个省（自治区）的２６个代表菌株及国外３个不同生理小种对照菌株进行ＲＡＰＤ－ＰＣＲ增,共产生了１４０个ＲＡＰＤ分子标记,其中８７．８％具有多态性。通过聚类分析确定了供试小种间的亲缘关系,并寻找到了我国３、７、８号小种的特异条带,为确立我国棉花枯萎菌生理小种在国际上的分类地位提供了可靠的分子证据。     

中国棉花枯萎菌生理小种的RAPD分析

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）分子标记方法对我国棉花枯萎菌３个生理小种（３、７、８号）进行遗传多样性分析,以筛选出的１０个随机引物对采自我国１１个省（自治区）的２６个代表菌株及国外３个不同生理小种对照菌株进行ＲＡＰＤ－ＰＣＲ增,共产生了１４０个ＲＡＰＤ分子标记,其中８７．８％具有多态性。通过聚类分析确定了供试小种间的亲缘关系,并寻找到了我国３、７、８号小种的特异条带,为确立我国棉花枯萎菌生理小种在国际上的分类地位提供了可靠的分子证据。     

Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population

Abstract The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium. Received: 29 April 1999; Accepted: 27 January 2000; Online Publication: 28 August 2000     

Use of ribotyping and random amplified polymorphic DNA to differentiate isolates of Burkholderia andropogonis

AIMS: The aim of this study was to determine the genetic diversity among isolates of Burkholderia andropogonis from various host plant species and geographic locations. METHODS AND RESULTS: Both random amplified polymorphic DNA (RAPD) and ribotyping analyses were used to assess the diversity of B. andropogonis isolates and compare these results with pathogenicity assays carried out on a number of common hosts of the organism. CONCLUSIONS: Both RAPD and ribotyping analyses revealed a high level of genetic diversity between isolates of B. andropogonis. Both methods demonstrated a similar clustering of isolates. However, there was no strict correlation between the genetic diversity revealed and the original host, geographic location or pathogenicity of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the genetic diversity of isolates of B. andropogonis. The great degree of diversity revealed in this study contrasts with the lack of phenotypic diversity within this species.     

Detection of genetic variability in various isolates of cattle tick, <Emphasis Type="Italic">Boophilus microplus</Emphasis> from Tamil Nadu,India using PCR-RAPD analysis

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

Genetic diversity in Hemileia vastatrix based on RAPD markers

Random amplified polymorphic DNA (RAPD) was used to assess the genetic structure of Hemileia vastatrix populations. Forty-five rust isolates with different virulence spectra and from different hosts and geographical regions were analyzed. Out of 45 bands, generated with three RAPD primers, 35 (78%) were polymorphic and scored as molecular markers. Cluster analysis exhibits unstructured variability of this pathogen with regard to physiological race, geographical origin or host. The genotypic diversity (H') inferred from Shannon's index was higher than gene diversity (Ht), suggesting that diversity is distributed among clonal lineages. Estimates of gene diversity in Africa and Asia populations were higher in total (Ht) as compared to within population diversity (Hs). Genetic differentiation was considerable among coffee rust isolates from Africa (Gst = 0.865) and Asia (Gst = 0.768) but not among isolates from South America (Gst = 0.266). We concluded that genetic diversity in H. vastatrix was moderately low and that the genetic differentiation among populations shows that asexual reproduction is likely to play an important role in the population biology of this fungus. This should be taken into account for the development of breeding programs.     

Characterization of Gibberella fujikuroi complex isolates by fumonisin B1 and B2 analysis and by RAPD and restriction analysis of PCR-amplified internal transcribed spacers of ribosomal DNA

Twenty nine isolates of Fusarium spp. (twenty four of them belonging to the Gibberella fujikuroi complex) isolated from banana and corn from different geographical regions were analyzed for their ability to produce fumonisins B1 and B2 and for genetic relatedness using random amplified polymorphic DNA (RAPD) and restriction analysis of PCR amplification products of the 5.8s ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II). For RAPD analysis, six of twenty oligonucleotide primers were selected after testing with five Fusarium spp. isolates and used to characterize 24 additional isolates. DNA fragments from the 29 isolates of Fusarium spp., which were approximately 560 bp, were amplified with the universal primers ITS1 and ITS4. The restriction enzymes HaeIII, MboI, HpaII and MspI were useful for distinguishing the isolates. The RAPD analysis permitted to find interspecific differences among the isolates of Fusarium spp., between isolates with low and high capacity of fumonisin production and among isolates from different hosts. The restriction fragment length polymorphism (RFLP-PCR) analysis permitted to distinguish among different species of Fusarium. In combination with morphological analysis, the results of this research may find an application for the diagnosis of unknown Fusarium spp. and, particularly, for the characterization of fumonisin-producing isolates, which may be very useful in the food technology field.     

Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.

We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.     

Characterization of Moroccan Isolates of Verticillium dahliaeKleb Using RAPD Markers

Isolates of Verticillium dahliae were sampled from different olive tree orchards in Morocco. These olive trees were located in different commercial culture locations in southern, central and northern Morocco. The isolates were characterized using genetic markers obtained after their DNA PCR amplification with random amplified polymorphic DNA (RAPD) primers. Among the 40 primers tested, 10 generated a total of 66 polymorphic fragments. Among the 38 isolates of V. dahliae tested, RAPD markers were successful in the characterization of groups based on their geographic origin. With the exception of one specific isolate, no correlation could be established among the isolates, based on the morphological appearance of the colony in culture.     

北方玉米丝轴黑粉菌遗传多样性与遗传分化研究

从80个随机扩增多态性DNA (RAPD)引物中筛选出26个扩增稳定、带纹清晰的引物,对从玉米丝黑穗病发生严重的大部分北方省市收集分离的68个玉米丝轴黑粉菌菌株进行了DNA遗传多 样性分析.结果表明,RAPD谱带多态性为98.33%,北方玉米丝轴黑粉菌具有丰富的遗传多样性,遗传分化明显;种群内遗传变异小于种群间遗传变异.玉米丝轴黑粉菌遗传分化除明显受地理阻隔影响外,也可能与玉米种子调运过程中携带病菌基因的遗传迁移相关.取相似系数为0.76阀值时的系统聚类分析,可将68个菌株划分为13个遗传宗谱.本研究结果完善和丰富了我国玉米丝轴黑粉菌遗传多样性评价研究,对玉米抗病资源筛选和抗病育种具有重要参考价值.